U.S. patent number 6,391,917 [Application Number 09/838,726] was granted by the patent office on 2002-05-21 for dialkyl ureas as calcitonin mimetics.
This patent grant is currently assigned to ZymoGenetics, Inc.. Invention is credited to Richard A. Houghten, Patricia A. McKernan, Jean-Philippe Meyer, Emma E. Moore, John M. Ostresh, Charles R. Petrie, Clemencia Pinilla.
United States Patent |
6,391,917 |
Petrie , et al. |
May 21, 2002 |
**Please see images for:
( Certificate of Correction ) ** |
Dialkyl ureas as calcitonin mimetics
Abstract
Dialkyl urea compounds are described which act as calcitonin
mimetics. These compounds are useful in the treatment of diseases
which are associated with bone resorption. The calcitonin mimetics
of the present invention are also useful in assays for the
determination of calcitonin receptor activity.
Inventors: |
Petrie; Charles R.
(Woodinville, WA), McKernan; Patricia A. (Woodinville,
WA), Moore; Emma E. (Seattle, WA), Ostresh; John M.
(Encinitas, CA), Meyer; Jean-Philippe (Holland, PA),
Houghten; Richard A. (Del Mar, CA), Pinilla; Clemencia
(Cardiff, CA) |
Assignee: |
ZymoGenetics, Inc. (Seattle,
WA)
|
Family
ID: |
22111012 |
Appl.
No.: |
09/838,726 |
Filed: |
April 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
410115 |
Sep 30, 1999 |
6255351 |
|
|
|
233893 |
Jan 20, 1999 |
6221913 |
Apr 24, 2001 |
|
|
Current U.S.
Class: |
514/596;
514/237.8; 514/255.06; 514/256; 514/419; 514/542; 514/564; 514/586;
514/878; 548/496; 548/516; 560/34; 562/439; 564/27; 564/47;
564/48 |
Current CPC
Class: |
A61P
19/08 (20180101); C07D 209/20 (20130101); C07C
275/28 (20130101); A61P 1/02 (20180101); A61P
5/18 (20180101); A61P 3/14 (20180101); A61P
19/10 (20180101); Y10S 514/878 (20130101) |
Current International
Class: |
C07C
275/28 (20060101); C07C 275/00 (20060101); C07D
209/20 (20060101); C07D 209/00 (20060101); A61K
031/17 (); A61K 031/404 (); A61K 031/496 (); A61K
031/50 (); A61K 031/537 () |
Field of
Search: |
;514/237.8,255.06,256,419,542,564,586,596,878 ;544/162,335,406
;548/496,516 ;560/34 ;562/439 ;564/27,47,48 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: O'Sullivan; Peter
Attorney, Agent or Firm: Lingenfelter; Susan E. Walsh; Brian
J.
Parent Case Text
This is a divisional application of application Ser. No.
09/410,115, filed Sep. 30, 1999, now U.S. Pat. No. 6,255,351, which
is a divisional of Ser. No. 09/233,893 filed on Jan. 20, 1999
issued U.S. Pat. No. 6,221,913 issued on Apr. 24, 2001, which
claims priority from application Ser. No. 60/072,987 filed on Jan.
21, 1998.
Claims
What is claimed is:
1. A method for providing an analgesic effect comprising
administering to a subject in need of such an effect an effective
amount of a calcitonin mimetic of formula I: ##STR53##
wherein
R1 and R2 are each members independently selected from the group
consisting of hydrogen, alkyls having from 1 to 6 carbon atoms,
alkenyls having from 1 to 6 carbon atoms, aryl, substituted aryl,
alkylaryl, substituted alkylaryl, carbocyclic ring, substituted
carbocyclic ring, heterocyclic ring, substituted heterocyclic ring,
and combinations thereof, the combinations are fused or covalently
linked and the substituents are selected from the group consisting
of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy,
haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl,
alkyl and aryl;
R3 is selected from the group consisting of hydrogen, aryl,
substituted aryl, carbocyclic ring, substituted carbocyclic ring,
heterocyclic ring, substituted heterocyclic ring, and combinations
thereof, the combinations are fused or covalently linked and the
substituents are selected from the group consisting of halogen,
haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino,
monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl;
R4 and R5 are each independently selected from the group consisting
of hydrogen and alkyls having from 1 to 6 carbon atoms, or taken
together from a ring selected from the group consisting of
saturated or unsaturated five-member rings, saturated or
unsaturated six-member rings and saturated or unsaturated
seven-member rings;
Z and X are each independently selected from the group NH, O, S, or
NR, wherein R is a lower alkyl group of from 1 to 6 carbon atoms;
and
n and m are each independently an integer from 0 to 6.
2. A method according to claim 1 wherein,
R1 is selected from the group consisting of phenyl, substituted
phenyl, benzyl, substituted benzyl, naphthylmethyl, substituted
naphthylmethyl, indolymethyl, and substituted indolymethyl;
R2 is selected from the group consisting of alkyls of from 1 to 6
carbon atoms, alkenyls having from 1 to 6 carbon atoms, benzyl,
substituted benzyl, naphthylmethyl, and substituted
naphthylmethyl;
wherein substituents are selected from the group consisting of
halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy,
haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl,
alkyl and aryl;
R4 and R5 are hydrogen;
Z is O; and
X is NH.
3. A method according to claim 2 wherein
R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl,
4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl,
4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl; and
R2 is ethyl, allyl, benzyl or 2-naphthylmethyl.
4. A method according to claim 1, wherein said calcitonin mimetic
has the formula: ##STR54##
wherein,
R1 and R2 are each independently selected from the group consisting
of hydrogen, alkyls having from 1 to 6 carbon atoms, alkenyls
having from 1 to 6 carbon atoms, aryl, substituted aryl, alkylaryl,
substituted alkylaryl, carbocyclic ring, substituted carbocyclic
ring, heterocyclic ring, substituted heterocyclic ring, and
combinations thereof, the combinations are fused or covalently
linked and the substituents are selected from the group consisting
of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy,
haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl,
alkyl and aryl; and
S1, S2, S3, S4 and S5 are each independently selected from the
group consisting of hydrogen, halogen, haloalkyl, hydroxy, aryloxy,
benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino,
acyloxy, acyl, alkyl and aryl.
5. A method according to claim 4 wherein,
R1 is selected from the group consisting of phenyl, substituted
phenyl, benzyl, substituted benzyl, naphthylmethyl, substituted
naphthylmethyl, indolymethyl, and substituted indolymethyl;
R2 is selected from the group consisting of alkyls of from 1 to 6
carbon atoms, alkenyls of from 1 to 6 carbon atoms, benzyl,
substituted benzyl, naphthylmethyl, and substituted
naphthylmethyl;
wherein the substituents are selected from the group consisting of
halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy,
haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl,
alkyl and aryl; and
S2 and S5 are t-butyl.
6. A method according to claim 5 wherein,
R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl,
4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl,
4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl;
R2 is ethyl, allyl, benzyl or 2-naphthylmethyl; and
S2 and S5 are t-butyl.
7. A method according to claim 1, wherein the analgesic effect
provides relief from bone pain.
Description
BACKGROUND OF THE INVENTION
Bone is a dynamic tissue, and homeostasis in the adult skeleton
requires a balance between bone resorption and bone formation.
Osteoclasts and osteoblasts play a key role in this balance, with
osteoclasts initiating bone resorption and osteoblasts synthesizing
and depositing new bone matrix. Imbalances in bone homeostasis are
associated with such conditions as osteoporosis, Paget's disease,
and hyperparathyroidism.
The activities of osteoclasts and osteoblasts are regulated by
complex interactions between systemic hormones and the local
production of growth factors and cytokines. Calcitonin, a peptide
hormone secreted by the thyroid and thymus of mammals, plays an
important role in maintaining bone homeostasis. Calcitonin inhibits
bone resorption through binding and activation of a specific
calcitonin receptor on osteoclasts (The Calcitonins-Physiology and
Pharmacology Azria (ed.), Karger, Basel, Su., 1989), with a
resultant decrease in the amount of calcium released by bone into
the serum. This inhibition of bone resorption has been exploited,
for instance, by using calcitonin as a treatment for osteoporosis,
a disease characterized by a decrease in the skeletal mass often
resulting in debilitating and painful fractures. Calcitonin is also
used in the treatment of Paget's disease where it provides rapid
relief from bone pain, which is frequently the primary symptom
associated with this disease. This analgesic effect has also been
demonstrated in patients with osteoporosis or metastatic bone
disease and has been reported to relieve pain associated with
diabetic neuropathy, cancer, migraine and post-hysterectomy.
Reduction in bone pain occurs before the reduction of bone
resorption.
Salmon calcitonin has been shown to be considerably more effective
in arresting bone resorption than human forms of calcitonin.
Several hypotheses have been offered to explain this observation:
1) salmon calcitonin is more resistant to degradation; 2) salmon
calcitonin has a lower metabolic clearance rate (MCR); and 3)
salmon calcitonin may have a slightly different conformation,
resulting in a higher affinity for bone receptor sites.
Despite the advantages associated with the use of salmon calcitonin
in humans, there are also disadvantages. For treatment of
osteoporosis, for instance, the average cost can exceed $75 a week
and involve daily prophylactic administration for 5 or more years.
In the United States, calcitonin must be administered by injection,
and since the disease indications for this drug are not usually
life threatening, patient compliance can be low. Resistance to
calcitonin therapy may occur with long-term use. What triggers this
resistance or "escape phenomenon" is unknown (see page 1093,
Principles of Bone Biology, Bilezikian et al., (eds.) Academic
Press, NY; Raisz et al., Am. J. Med. 43:684-90, 1967; McLeod and
Raisz, Endocrine Res. Comm.8:49-59, 1981; Wener et al.,
Endocrinology. 90:752-9, 1972 and Tashjian et al., Recent Prog.
Horm. Res. 34:285-303, 1978). Use of calcitonin mimetics, either in
place of native calcitonins or in rotation with native calcitonins,
would help avoid resistance to such treatment during long-term use.
In addition, some patients develop antibodies to non-human
calcitonin, calcitonin mimetics would be useful for such
patients.
What is needed in the art are alternative methods of inhibiting
bone resorption. The present invention fulfills these and other
needs.
SUMMARY OF THE INVENTION
The present invention provides isolated compounds that are useful
as calcitonin mimetics. As used herein, the term "calcitonin
mimetic" refers to a compound with the ability to mimic the effects
generated by calcitonin's interaction with its receptor and its
signal transduction pathway and, by such interaction, stimulate
G-protein-mediated activation by adenyl cyclase.
Within one aspect the invention provides a compound of formula I:
##STR1##
wherein R1 and R2 are each members independently selected from the
group consisting of hydrogen, alkyls having from 1 to 6 carbon
atoms, alkenyls having from 1 to 6 carbon atoms, aryl, substituted
aryl, alkylaryl, substituted alkylaryl, carbocyclic ring,
substituted carbocyclic ring, heterocyclic ring, substituted
heterocyclic ring, and combinations thereof, the combinations are
fused or covalently linked and the substituents are selected from
the group consisting of halogen, haloalkyl, hydroxy, aryloxy,
benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino,
acyloxy, acyl, alkyl and aryl; R3 is a 2,5 disubstituted aryl; R4
and R5 are each independently selected from the group consisting of
hydrogen and alkyls having from 1 to 6 carbon atoms, or taken
together from a ring selected from the group consisting of
saturated or unsaturated five-member rings, saturated or
unsaturated six-member rings and saturated or unsaturated
seven-member rings; Z and X are each independently selected from
the group NH, O, S, or NR, wherein R is a lower alkyl group of from
1 to 6 carbon atoms; n and m are each independently an integer from
0 to 6. Within one embodiment R1 is selected from the group
consisting of phenyl, substituted phenyl, benzyl, substituted
benzyl, naphthylmethyl, substituted naphthylmethyl, indolymethyl,
and substituted indolymethyl; R2 is selected from the group
consisting of alkyls of from 1 to 6 carbon atoms, alkenyls of from
1 to 6 carbon atoms, benzyl, substituted benzyl, naphthylmethyl,
and substituted naphthylmethyl; wherein substituents are selected
from the group consisting of halogen, haloalkyl, hydroxy, aryloxy,
benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino,
acyloxy, acyl, alkyl and aryl; and R4 and R5 are hydrogen; Z is O;
and X is NH. Within a related embodiment R1 is 4-ethoxybenzyl,
1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl,
1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl,
2-naphthylmethyl or phenyl; and R2 is ethyl, allyl, benzyl or
2-naphthylmethyl. Within another embodiment the compound has the
formula: ##STR2##
wherein, R1 and R2 are each independently selected from the group
consisting of hydrogen, alkyls having from 1 to 6 carbon atoms,
alkenyls having from 1 to 6 carbon atoms, aryl, substituted aryl,
alkylaryl, substituted alkylaryl, carbocyclic ring, substituted
carbocyclic ring, heterocyclic ring, substituted heterocyclic ring,
and combinations thereof, the combinations are fused or covalently
linked and the substituents are selected from the group consisting
of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy,
haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl,
alkyl and aryl; and S1, S3 and S4 are each independently selected
from the group consisting of hydrogen, halogen, haloalkyl, hydroxy,
aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino,
dialkylamino, acyloxy, acyl, alkyl and aryl. S2 and S5 are each
independently alkyl or aryl. Within one embodiment R1 is selected
from the group consisting of phenyl, substituted phenyl, benzyl,
substituted benzyl, naphthylmethyl, substituted naphthylmethyl,
indolymethyl, and substituted indolymethyl; R2 is selected from the
group consisting of alkyls having from 1 to 6 carbon atoms,
alkenyls having from 1 to 6 carbon atoms, benzyl, substituted
benzyl, naphthylmethyl, and substituted naphthylmethyl; wherein the
substituents are selected from the group consisting of halogen,
haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino,
monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl and S2
and S5 are t-butyl. Within a related embodiment R1 is
4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl,
1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl,
2-naphthylmethyl or phenyl; R2 is ethyl, allyl, benzyl or
2-naphthylmethyl; and S2 and S5 are t-butyl.
Within another aspect, the invention provides a pharmaceutical
composition comprising an effective amount of a compound as
described above in a pharmaceutically acceptable carrier.
Within another aspect the invention provides a method for treating
a bone-related disorder, comprising administering to a subject
suffering from such disorder an effective amount of calcitonin
mimetic compound as described above. Within a related embodiment
the bone-related disorder is selected from the group consisting of
osteoporosis, Paget's disease, hyperparathyroidism, osteomalacia,
periodontal applications (bone loss), hypercalcemia of malignancy
and hypercalcemia of infancy.
Within another aspect the invention provides a method of inhibiting
bone resorption comprising administering to a subject in need of
such inhibition an effective amount of a calcitonin mimetic
compound as described above.
Within yet another aspect the invention provides a method for
providing an analgesic effect comprising administering to a subject
in need of such an effect an effective amount of a calcitonin
mimetic compound as described above. Within a related embodiment
the analgesic effect provides relief from bone pain.
Within another aspect the invention provides a method for treating
conditions associated with inhibiting gastric secretion comprising
administering to a subject in need of such inhibition an effective
amount of a calcitonin mimetic compound as described above. Within
a related embodiment the conditions associated with inhibiting
gastric secretion is a gastrointestinal disorder.
These and other aspects of the invention will become evident upon
reference to the following detailed description and the attached
drawings.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations
The following abbreviations are used herein: Boc, t-butoxycarbonyl;
DCM, dichloromethane; DME, dimethoxyethane; DMF, dimethylformamide;
EtOAc, ethyl acetate; Fmoc, fluorenylmethoxycarbonyl; TFA,
trifluoroacetic acid.
All references cited herein are incorporated by reference in their
entirety.
The calcitonin mimetics which are useful in the present invention
are those compounds with the ability to mimic the interaction of
calcitonin with its receptor and, by such mimicry, to stimulate
G-protein-mediated activation of adenyl cyclase or activation of
CRE by an alternative signal transduction pathway. These mimetics
are represented by the general formula: ##STR3##
In this formula, R1 and R2 are each independently hydrogen, alkyl
groups having from 1 to 6 carbon atoms, alkenyl groups having from
1 to 6 carbon atoms, an aryl group, or alkylaryl groups, where the
alkyl portion may have 1 to 6 carbon atoms and the aryl portion
represents an aryl group, a substituted aryl group, a carbocyclic
ring, a substituted carbocyclic ring, a heterocyclic ring, a
substituted heterocyclic ring, or combinations thereof. The
combinations can be fused or covalently linked. In certain
preferred embodiments R1 is substituted or unsubstituted phenyl,
benzyl, naphthylmethyl or indolymethyl. R2 is an alkyl or alkenyl
having from 1 to 6 carbon atoms, substituted or unsubstituted
benzyl or naphthylmethyl. In certain particularly preferred
embodiments R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl,
4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl,
4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl. R2 is
ethyl, allyl, benzyl, or 2-naphthylmethyl.
R3 represents substituted and unsubstituted aryl groups,
carbocyclic rings, heterocyclic rings, or combinations thereof. The
combinations can be fused or covalently linked. Within certain
preferred embodiments R3 is a 2,5 disubstituted aryl. Preferably
the substitutions are aryl or alkyl. Within a preferred embodiment
R3 is 2,6-di-t-butyl-phenyl.
R4 and R5 are each independently hydrogen, alkyl groups having from
1 to 6 carbon atoms. Within certain embodiments R4 and R5 can be
joined together to form a ring which is a four-, five-, six- or
seven-member ring, saturated or unsaturated. For those embodiments
in which the ring is unsaturated, the ring can be an heteroaromatic
ring (e.g., pyrimidyl, imidazyl). Within certain preferred
embodiments R4 and R5 are hydrogen.
Z and X each independently represent either NH, NR, O, or S, in
which R is a lower alkyl group of from one to six carbon atoms. In
preferred embodiments, Z represents O and X represents NH. The
symbols n and m each represent independently, integers from zero to
six.
As used herein, the term "alkyl" refers to a saturated hydrocarbon
radical which may be straight-chain or branched-chain (for example,
ethyl, isopropyl, or t-butyl), or cyclic (for example cyclobutyl,
cyclopropyl or cyclopentyl). Preferred alkyl groups are those
containing 1 to 6 carbon atoms.
The term "alkenyl" refers to an unsaturated hydrocarbon radical
which may be a straight-chain, branched-chain or cyclic. Examples
of alkenyls include vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl,
3-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl
and 5-hexenyl, as well as dienes and trienes of straight, branched
or cyclic chains and the like. Preferred alkenyl groups are those
containing 1 to 6 carbon atoms.
The term "aryl" refers to an aromatic substituent which may be a
single ring or multiple rings which are fused together, linked
covalently or linked to a common group such as an ethylene or
methylene moiety. The aromatic rings may each contain heteroatoms,
preferred heteroatoms are N, S or O. Examples of aryl groups
include phenyl, benzyl, naphthyl, biphenyl, diphenylmethyl,
2,2-diphenyl-1-ethyl, thienyl, pyridyl and quinoxalyl.
Additionally, the aryl groups may be attached to other parts of the
molecule at any position on the aryl radical which would otherwise
be occupied by a hydrogen atom (such as, for example, 2-pyridyl,
3-pyridyl and 4-pyridyl).
Heterocyclic rings contain at least one heteroatom selected from N,
O and S. Examples of carbocyclic and heterocyclic rings include
cyclohexyl, cyclohexenyl, piperazinyl, pyrazinyl, morpholinyl,
imidazolyl, triazolyl and thiazolyl.
The terms "substituted alkyl", "substituted alkenyl", "substituted
alkylaryl", "substituted aryl", "substituted carbocyclic ring",
"substituted heterocyclic ring" "substituted phenyl", "substituted
benzyl", "substituted naphthylmethyl" and "substituted
indolymethyl" refer to the above alkyl, alkenyl, alkylaryl,
carbocyclic ring, heterocyclic ring, aryl, phenyl, benzyl,
naphthylmethyl and indolymethyl groups substituted by one or more,
preferably one, halogen, haloalkyl, hydroxy, aryloxy, benzyloxy,
alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy,
acyl, alkyl and aryl. Examples include 4-ethoxybenzyl,
1-ethyl-indolylmethyl, 4-alloxybenzyl, 1-allyl-indolylmethyl,
4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl or
2-naphthylmethyl.
All numerical ranges in this specification and claims are intended
to be inclusive of their upper and lower limits.
In one group of preferred embodiments, the calcitonin mimetics are
represented by the formula: ##STR4##
In this formula, the symbols R1 and R2 have the meaning provided
above. The symbols S1, S3 and S4 each independently represent a
substituent on the attached aromatic ring which is hydrogen,
halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy,
haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl,
alkyl and aryl. The symbols S2 and S3 each represent an aryl or
alkyl. In certain preferred embodiments R1 is substituted or
unsubstituted phenyl, benzyl, naphthylmethyl or indolymethyl. R2 is
an alkyl or alkenyl having from 1 to 6 carbon atoms, substituted or
unsubstituted benzyl or naphthylmethyl. In certain particularly
preferred embodiments, S5 and S2 are t-butyl, R1 is 4-ethoxybenzyl,
1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl,
1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl,
2-naphthylmethyl or phenyl and R2 is ethyl, allyl, benzyl, or
2-naphthylmethyl.
The calcitonin mimetics used in the present invention can be
prepared using commercially available materials. A general
synthetic scheme for preparing molecules of Formula I wherein R4
and R5 are hydrogen, using methodologies known in the art, is
provided herein. ##STR5##
In a typical preparation, 100 mg of p-methylbenzhydrylamine (MBHA)
resin (0.81 meq/g, 100-200 mesh) was contained within a sealed
polypropylene mesh packet. Following neutralization with 5%
diisopropylethylamine (DIEA) in dichloromethane (DCM), the resin
was washed with DCM. The first protected amino acid was coupled
using hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide
(DICI) in DMF. Following removal of the amino protecting group, the
mesh packet was shaken overnight in a solution of 0.1 M trityl
chloride in DCM/DMF (9:1) in the presence of DIEA. Completeness of
the trityl coupling was verified using the bromophenol blue color
test as described in Krchnak et al., (Coll. Czech. Chem. Commun.
53:2542, 1988, and repeated as necessary.
N-alkylation was then performed by treatment of the resin packet
with 1 M lithium t-butoxide in THF (20.times.) for 15 min, as
described by Drner, et al., (Bioorg. Med. Chem. 4:709, 1996).
Excess base was then removed by decantation, followed by addition
of the individual alkylating agent in DMSO (20.times., 0.1M). The
solution was vigorously shaken for 2 h at room temperature. This
step is normally repeated three times for methyl iodide, and five
times for the other alkylating agents. Small aliquots of the resin
can be cleaved to determine the completeness of this step. The
trityl group was removed with 2% TFA in DCM (2.times.10 min).
The isocyanate of the incoming primary amine (or aniline) was
performed by slowly adding a solution of the primary amine (0.3M in
DCM, 24.times. over the resin substitution) and DIEA (48.times.)
dropwise to solution of 0.1M triphosgene (8.times.) in DCM. It is
known in the art that the reaction does not proceed through the
isocyanate for secondary amines. The packet was washed, neutralized
and the isocyanate solution added and shaken for 1 hour at RT.
Following decantation, the isocyanate solution was quenched with
10% NH.sub.3 in DMF. The resin was washed with DCM, 0.05% NH.sub.3
in DMF, MeOH, DCM, and MeOH.
The product was cleaved from the resin with anhydrous HF by the
procedures of Houghten et al., (Int. J. Pep. Prot. Res. 27:673,
1986), in the presence of anisole. The product was extracted with
50% ACN/H.sub.2 O and lyophilized, followed by relyophilization
from 50% acetonitrile.
The compounds of the invention can be administered to warm blooded
animals, including humans, to mimic the interaction of calcitonin
with its receptor in vivo. Within one aspect, calcitonin mimetics
of the present invention are contemplated to be advantageous for
use in therapeutic defects for which calcitonin is useful. In
particular, the calcitonin mimetics are useful for the regulation
of bone metabolism and reduction of serum calcium. The calcitonin
mimetics of the invention can be administered to warm blooded
animals, including humans, to mimic the interaction of calcitonin
with its receptor in vivo. Thus, the present invention encompasses
methods for therapeutic treatment of bone-related disorders. Such
bone-related disorders include, but are not limited to,
osteoporosis, Paget's Disease, hyperparathyroidism, osteomalacia,
periodontal defects (bone loss), hypercalcemia of malignancy,
idiopathic hypercalcemia of infancy, and other related conditions.
Calcitonin mimetics are also contemplated to be advantageous as
analgesics, in particular for relief of bone pain. Calcitonin
mimetics are further contemplated to be advantageous in inhibiting
bone resorption. The calcitonin mimetics of the present invention
can also be used to inhibit gastric secretion in the treatment of
acute pancreatitis and gastrointestinal disorders. The methods of
the present invention may be used to treat these conditions in
their acute or chronic stages.
Pharmaceutically or therapeutically effective amounts of calcitonin
mimetics of the present invention can be formulated with
pharmaceutically or therapeutically acceptable carriers for
parenteral, oral, nasal, rectal, topical, transdermal
administration or the like, according to conventional methods.
Formulations may further include one or more diluents, fillers,
emulsifiers, preservatives, buffers, excipients, and the like, and
maybe provided in such forms as liquids, powders, emulsions,
suppositories, liposomes, transdermal patches and tablets, for
example. Slow or extended-release delivery systems, including any
of a number of biopolymers (biological-based systems), systems
employing liposomes, and polymeric delivery systems, can also be
utilized with the compositions described herein to provide a
continuous or long-term source of the calcitonin mimetic. Such slow
release systems are applicable to formulations, for example, for
oral, topical and parenteral use. The term "pharmaceutically or
therapeutically acceptable carrier" refers to a carrier medium
which does not interfere with the effectiveness of the biological
activity of the active ingredients and which is not toxic to the
host or patient. One skilled in the art may formulate the compounds
of the present invention in an appropriate manner, and in
accordance with accepted practices, such as those disclosed in
Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack
Publishing Co., Easton, Pa., 19th ed., 1995. Preferably such
compounds would be administered orally or parenterally.
As used herein, a "pharmaceutically or therapeutically effective
amount" of such a calcitonin mimetic is an amount sufficient to
induce a desired biological result. The result can be alleviation
of the signs, symptoms, or causes of a disease, or any other
desired alteration of a biological system. For example, an
effective amount of a calcitonin mimetic is that which provides
either subject relief of symptoms or an objectively identifiable
improvement as noted by the clinician or other qualified observer.
In particular, such an effective amount of a calcitonin mimetic
results in reduction in serum calcium, inhibition of bone
resorption, inhibition of gastric secretion or other beneficial
effect. Effective amounts of the calcitonin mimetics can vary
widely depending on the disease or symptom to be treated. The
amount of the mimetic to be administered, and its concentration in
the formulations, depends upon the vehicle selected, route of
administration, the potency of the particular mimetic, the clinical
condition of the patient, the side effects and the stability of the
compound in the formulation. Thus, the clinician will employ the
appropriate preparation containing the appropriate concentration in
the formulation, as well as the amount of formulation administered,
depending upon clinical experience with the patient in question or
with similar patients. Such amounts will depend, in part, on the
particular condition to be treated, age, weight, and general health
of the patient, and other factors evident to those skilled in the
art. Estimation of appropriate dosages effective for the individual
patient is well within the skill of the ordinary prescribing
physician or other appropriate health care practitioner. As a
guide, the clinician can use conventionally available advice from a
source such as the Physician's Desk Reference, 48.sup.th Edition,
Medical Economics Data Production Co., Montvale, N.J. 07645-1742
(1994). Typically a dose will be in the range of 0.1-100 mg/kg of
subject. Preferably 0.5-50 mg/kg. Doses for specific compounds may
be determined from in vitro or ex vivo studies on experimental
animals. Concentrations of compounds found to be effective in vitro
or ex vivo provide guidance for animal studies, wherein doses are
calculated to provide similar concentrations at the site of
action.
Well established animal models are available to test in vivo
efficacy of calcitonin mimetics. For example, the hypocalcemic rat
model can be used to determine the effect of synthetic calcitonin
mimetics on serum calcium, and the ovariectomized rat or mouse can
be used as a model system for osteoporosis. Bone changes seen in
these models and in humans during the early stages of estrogen
deficiency are qualitatively similar. Calcitonin has been shown to
be an effective agent for the prevention of bone loss in
ovariectomized humans and also in rats (Mazzuoli, et al., Calcif.
Tissue Int. 47:209-14, 1990; Wronski, et al., Endocrinology
129:2246-50, 1991).
Only those compounds which retain calcitonin-like activity, as
assayed by a CRE-luciferase assay, for example, are within the
scope of this invention. The calcitonin receptor is a member of the
G-protein receptor family and transduces signal via activation of
adenylate cyclase, leading to elevation of cellular cAMP levels
(Lin, et al., Science 254:1022-4, 1991). This assay system exploits
the receptor's ability to detect other molecules, not calcitonin,
that are able to stimulate the calcitonin receptor and initiate
signal transduction.
Receptor activation can be detected by: (1) measurement of
adenylate cyclase activity (Salomon, et al., Anal. Biochem.
58:541-8, 1974; Alvarez and Daniels, Anal. Biochem. 187:98-103,
1990); (2) measurement of change in intracellular cAMP levels using
conventional radioimmunoassay methods (Steiner, et al., J. Biol.
Chem. 247:1106-13, 1972; Harper and Brooker, J. Cyc. Nucl. Res.
1:207-18, 1975); or (3) use of a CAMP scintillation proximity assay
(SPA) method (Amersham Corp., Arlington Heights, Ill.). While these
methods provide sensitivity and accuracy, they involve considerable
sample processing prior to assay, are time consuming, may involve
the use of radioisotopes, and would be cumbersome for large scale
screening assays.
An alternative assay system (described in WO96/31536) involves
selection of substances that are able to induce expression of a
cyclic AMP response element (CRE)-luciferase reporter gene, as a
consequence of elevated cAMP levels or other signaling pathways,
such as stimulation of Ca.sup.++ /Ip.sub.3 pathway leading to CRE
induction, in cells expressing a calcitonin receptor, but not in
cells lacking calcitonin receptor expression. Such cells could
include, for example, Boris/KS10-3 (expressing hamster calcitonin
receptor and a CRE-luciferase reporter gene in baby hamster kidney
cells (BHK 570 cells)) or Hollex 1 or Hollex 2 (expressing human
calcitonin receptor and a CRE-luciferase reporter gene in BHK
cells, as described in WO96/31536) or KZ10-20-48/pLJ6-4-25, which
expresses the human glucagon receptor and a CRE-luciferase reporter
gene in BHK cells. The human glucagon receptor is another member of
the G-protein-coupled receptor that transduces signal through
adenylate cyclase-mediated elevation of cAMP. PTH can be used as a
control as well.
This CRE-luciferase assay measures the end result of a multi-step
signal transduction pathway triggered when a calcitonin mimetic
stimulates the G-coupled calcitonin receptor. The complexity of
this pathway provides multiple mechanisms for induction of
luciferase transcription at points that are downstream of the
calcitonin receptor, and therefore may not be calcitonin
receptor-specific (e.g., forskolin's direct activation of adenylate
cyclase). Any response triggered by non-specific inducers is
eliminated by counter screening using the calcitonin
receptor-negative cell lines described above.
The foregoing description and the following examples are offered
primarily for illustration and not as limitations. It will be
readily apparent to those of ordinary skill in the art that the
operating conditions, materials, procedural steps and other
parameters of the system described herein may be further modified
or substituted in various ways without departing from the spirit
and scope of the invention. The invention is further illustrated by
the following non-limiting examples.
EXAMPLES
Example 1
Preparation of Calcitonin Mimetics: Urea of Aniline and L-Leucine
Methylamide
Preparation of Trityl-leucine Amide Resin
In the preparation of the urea of aniline and leucine methyl amide,
100 mg of p-methylbenzhydrylamine (MBHA) resin (0.81 meq/g, 100-200
mesh) was contained within a sealed polypropylene mesh packet as
used in simultaneous multiple synthesis as described in Houghten,
(Proc. Natl. Acad. Sci. USA 82:5131, 1985). Following
neutralization (1 min) with 5% diisopropylethylamine (DIEA) in
dichloromethane (DCM) (3.times.5 ml), the resin was washed with DCM
(3.times.5 ml). The resin packet was added to a solution of
tert-butyloxycarbonyl-L-leucine (Boc-Leu) (111 mg, 0.48 meq) and
1-hydroxybenzotriazole (65 mg, 0.48 meq) in dimethylformamide (DMF)
(2.4 ml) in a 10 ml polypropylene bottle. Following addition of 2.4
ml 0.2 M diisopropylcarbodiimide (DICI) in DMF, the resin was
shaken on a reciprocation shaker for 1.5 h. The resin was then
washed with DMF (3.times.5 ml) and DCM (3.times.5 ml). The Boc
protecting group was then removed by treatment with 55%
trifluoroacetic acid in DCM for 30 min. The resin was then washed
with DCM (2.times.5 ml), isopropanol (IPA, 3.times.5 ml), DCM
(3.times.5 ml), neutralized with 5% DIEA in DCM (3.times.5 ml), and
washed with DCM (2.times.5 ml). The resin packet was then shaken
overnight (16 h) in 5 ml of 0.1M trityl chloride in DCM/DMF (9:1)
in the presence of DIEA. Completeness of the trityl coupling was
verified using the bromophenol blue color test as described in
Krchnak et al., (Coll. Czech. Chem. Commun. 53:2542, 1988), and
repeated as necessary. N-Methylation of trityl-leucine amide
resin
N-methylation was then performed by treatment of the resin packet
with a solution of 3.2 ml 0.5 M lithium t-butoxide (LiOtBu) in THF
for 15 min, as described by Drner, et al., (Bioorg. Med. Chem.
4:709, 1996). Excess base was then removed by decantation, followed
by addition of a solution of 0.3 ml methyl iodide in 3.2 ml
dimethylsulfoxide (DMSO). The solution was shaken for 2 hours at
room temperature. The solution was then removed, washed with THF
(1.times.5 ml) and the LiOtBu/methyl iodide treatment repeated.
Following removal of the solution, the resin was washed with DMF
(3.times.5 ml), IPA (2.times.5 ml), DCM (3.times.5 ml). The trityl
group was removed by two treatments (10 min) with 2% TFA in DCM (5
ml). The resin was then washed with DCM (2.times.5 ml), IPA
(3.times.5 ml) and DCM (3.times.5 ml).
Preparation of the Urea of Aniline and Leucine Methylamide
Resin
The resin packet was neutralized (1 min) with 5% DIEA in DCM
(3.times.5 ml), and washed with DCM (2.times.5 ml). The isocyanate
of aniline was then performed by slowly adding a solution of
aniline (0.176 ml) in DCM (6.5 ml) and DIEA (0.678 ml) dropwise
with stirring to a solution of triphosgene (193 mg) in DCM (6.5
ml). The resin packet was added to the isocyanate solution and
shaken for 1 hour at room temperature. Following decantation, the
resin was washed with DCM (1.times.5 ml), 0.05% NH3 in DMF
(2.times.5 ml), IPA (1.times.5 ml), DCM (1.times.5 ml), and
methanol (1.times.5 ml). The resin was then dried under high vacuum
overnight.
Cleavage of the Urea from the Resin
The product was cleaved from the resin using 5 ml of anhydrous HF
for 1.5 h at 0.degree. C. by the procedures of Houghten et al.,
(Int. J. Pep. Prot. Res. 27:673, 1986). The product was extracted
with 50% acetonitrile(ACN)/H.sub.2 O (2.times.5 ml) and
lyophilized, followed by relyophilization from 50% ACN/H.sub.2 O (5
ml). 13.5 mg crude product (85% purity by RP-HPLC) having the
expected molecular weight of 263 daltons was obtained.
Example 2
This example provides in vitro, ex vivo, and in vivo assays which
can be used to evaluate compounds described herein for their use in
therapeutic applications.
Assays for Calcitonin Mimetic Activity
CRE-Luciferase Assay Method for Calcitonin Mimetics
Human calcitonin receptor-positive and receptor-negative BHK-570
(Baby Hamster Kidney) cell lines were maintained by serial passage
in growth medium (DMEM supplemented with 10% heat-inactivated fetal
calf serum (HI-FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, 250 nM
MTX, and 1 mg/mL G418). On the day prior to assay, cells were
trypsinized, adjusted to 2.5.times.10.sup.5 cells/ml in growth
medium, plated in opaque white Dynatech Microlite microtiter tissue
culture plates at 50 .mu.L/well, and grown overnight to confluence
(37.degree. C., 5% CO.sub.2 atmosphere).
Test substances were prepared in DMSO or 10% DMF at 100 times the
final desired assay concentration. At the time of assay, test
substances were diluted into assay medium to 100, 50, 25, and 12.5
.mu.g/ml in DMEM supplemented with 10% HI-FCS, 2 mM L-glutamine, 1
mM sodium pyruvate and 20 mM Hepes, pH 7.25, then 50 .mu.l/well was
added to assay plates for final assay concentrations of 50, 25,
12.5 and 6.25 .mu.g/ml in 1% DMSO (or 0.1% DMF). Controls were
included on each plate: untreated wells (basal), 25 mM forskolin,
and 100 nM human calcitonin. DMSO or DMF was included in control
wells at a concentration equal to that in test samples (not to
exceed a final assay concentration of 2% DMSO or 0.5% DMF, with a
preferred maximum of 1% DMSO or 0.1% DMF).
Plates were incubated for 3 to 8 hours (4 hours preferred) at
37.degree. C. in an atmosphere of 5% CO.sub.2. Following induction,
luciferase activity was measured using a Promega luciferase assay
kit (E1500) according to the assay kit protocol (Promega Corp.,
Madison, Wis.). Briefly, assay medium was removed and cells were
washed once with phosphate buffered saline (PBS). After the wash,
25 .mu.l of lysis buffer was added to each well, and the plates
were incubated for 15 minutes at room temperature. Fifty
microliters of Luciferase Assay Substrate (Promega, Corp.) was
added to each well and the plates were transferred to a Labsystems
Lumiscan microtiter luminometer (Labsystems Inc., Morton Grove,
Ill.). The amount of luminescence (relative light units, RLU) was
determined following a 0.1 second/well integration of signal. Basal
(uninduced) luciferase signal was subtracted from all measurements,
and the luciferase signal induced by test samples was expressed as
a percentage of the signal in the calcitonin and forskolin
controls. Specificity of the luciferase induction for calcitonin
receptor-positive cell lines was determined by comparing the
percent control values in the calcitonin receptor-positive line
(Hollex-1) to those observed in the calcitonin receptor-negative
cell line (KZ10-20-48/Zem 228) and the PTH receptor-positive cell
line (KZ10-20-48/PTH-20) described below. Samples inducing a signal
over the basal level were selected for further characterization,
see Table 1 for examples.
TABLE 1 Luciferase induction (% of maximum luciferase induction
produced by CT, 100 nM) ##STR6## Crude .mu.g/ml TPI # R1 R2 50 25
12.5 6.25 3.125 628-007 ##STR7## ##STR8## 31.73 55.34 31.97 10.61
0.76 628-008 ##STR9## ##STR10## 50.39 50.93 20.07 2.91 -1.31
628-013 ##STR11## ##STR12## 64.26 69.62 51.62 17.98 3.51 628-015
##STR13## ##STR14## 71.49 72.17 43.2 14.69 1.62 628-016 ##STR15##
##STR16## 75.76 80.63 76.44 43.89 10.72 628-022 ##STR17## ##STR18##
46.98 41.52 17.85 3.26 2.08 628-023 ##STR19## ##STR20## 41.79 46.84
32.04 7.84 -0.46 628-024 ##STR21## ##STR22## 30.25 22.64 11.8 1.39
-1.24 628-025 ##STR23## ##STR24## 22.61 7.96 2.33 0.65 0.42 628-042
##STR25## ##STR26## 60.41 68.69 59.74 49.17 19.16 628-043 ##STR27##
##STR28## 47.95 46.24 42.06 34.53 6.48 628-044 ##STR29## ##STR30##
30.14 26.23 29.35 24.08 10.65 628-045 ##STR31## ##STR32## 40.62
44.51 42.49 38.73 20.19 628-052 ##STR33## ##STR34## 38.27 40.21
36.63 23.59 9.57 628-053 ##STR35## ##STR36## 40.14 41.8 32.31 11.89
3.29 628-054 ##STR37## ##STR38## 54.86 47.47 42.54 27.05 10.73
628-056 ##STR39## ##STR40## 37.1 35.6 28.6 14.7 4.2 628-055
##STR41## ##STR42## 48.69 43.3 35.48 24.81 628-032 ##STR43##
##STR44## 33.94 31.34 27.28 25.42 628-033 ##STR45## ##STR46## 33.62
31.63 27.68 14.71 628-034 ##STR47## ##STR48## 38.68 33.86 25.02
6.89 628-035 ##STR49## ##STR50## 52.27 46.43 43.25 34.84 628-036
##STR51## ##STR52## 10.38 8.0 6.43 4.12
Test substances that appear to specifically elevate luciferase
expression in CT-R positive cells but not CT-R negative cells were
subjected to an additional specificity check, i.e. their inability
to activate other members of the G-protein coupled receptor family.
The parathyroid hormone (PTH) receptor is another member of the
G-protein coupled receptor family that transduces signal through
adenylate cyclase mediated elevation of cAMP. The receptor negative
CRE-luciferase/DHFR expressing BHK570 clone (KZ10-20-48) was
transfected with the plasmid phupthr.2, encoding the cloned human
PTH receptor in plasmid pHZ-1 which also contains the G418
selectable marker. Stable transfectants were selected in 250 nM
MTX+1 mg/ml G418 and were screened for CRE-luciferase induction in
response to 25 mM forskolin or 100 nM human PTH (Sigma) (as
described in WO96/31536). Clone KZ10-20-48/PTH-20 was selected for
use in specificity confirmation. This clone exhibits a 25 fold
induction of luciferase in response to human PTH (EC50=0.02 nM ) or
forskolin (EC50=2.0 uM).
Calvarial Assay
Calvaria from 4-day old neonatal CD-1 mice (pregnant mice received
from Charles River Laboratories, Wilmington, Mass.) were trimmed
with fine-tipped scissors to leave the parietal regions, including
the sagittal suture. These trimmed bones were placed singly per
well into 6-well cell culture cluster plates (Costar, Pleasanton,
Calif.) with 1 ml/well of Dulbecco's Minimum Essential Medium, 4.5
ug/ml glucose (DMEM, BioWhittaker, Walkersville, Md.) or Basal
Eagle's Medium with Earle's salts (EMEM, Gibco/BRL, Grand Island,
N.Y.) and 0.29 mg/ml L-glutamine, 1 mM sodium pyruvate, 15%
heat-inactivated horse serum, and antibiotics (penicillin-G 50
.mu.g/ml, streptomycin 50 .mu.g/ml, and neomycin 100 .mu.g/ml).
Calvaria were rocked gently (RedRocker.TM., model PR50-115 V,
Hoefer, San Francisco, Calif. or Labline Rocking Shaker, model
4635, Labline Instruments, Melrose Park, Ill.) at 37.degree. C. in
a 5% CO.sub.2 humidified incubator for 24 hours preincubation.
Following preincubation, medium was removed and replaced with 1.5
ml/well of growth medium containing 1 nM parathyroid hormone (PTH)
1-34 (Sigma) to stimulate bone resorption. For evaluation of the
ability of calcitonin mimetics to inhibit PTH induced bone
resorption, mimetic compounds in DMSO were added to the growth
medium at concentrations ranging from 1-400 .mu.g/ml (final assay
concentration of DMSO less than or equal to 1%). In each experiment
human calcitonin (0.02-2 nM, 0.2-2 nM preferred) was added to PTH
treated bones as a positive control. Control wells that did not
receive PTH, human calcitonin or calcitonin mimetic were included
for determination of calcium release from untreated bones. All
control wells contained a final assay concentration of DMSO equal
to that present in calcitonin mimetic treated wells.
Five bones were included in each sample group. Bones were incubated
for 72 hours following PTH addition to allow resorption of bone to
occur. Observations were made of the general appearance,
healthiness and number of cells. that migrate from the calvaria
during the incubation as a possible indication of cell toxicity.
Calvaria to be examined histologically were transferred to glass
scintillation vials containing 10 ml of 10% neutral buffered
formalin.
The medium was removed from the wells, and total calcium
measurements were made using a Nova 7/7+7 Electrolyte Analyzer or
Nova CRT 10 analyzer (Nova Biomedical, Waltham, Mass.) according to
the manufacturer's specifications. Induction of bone resorption by
PTH is seen as an increase in the concentration of calcium in the
growth medium due to degradation of the bone matrix. Human
calcitonin and biologically active calcitonin mimetics inhibit this
bone resorptive process as demonstrated by a lowering of the
calcium in growth medium as compared to bones treated with PTH
alone.
Calvaria Histology
To confirm the findings in the calvarial bone resorption assay
employing calcium release from culture mouse calvariae, selected
bones were fixed in 10% neutral buffered formalin and demineralized
in 5% formic acid with 5% formalin. The bones were dehydrated
through an ascending series of ethanol concentrations, infiltrated
in glycol methacrylate, and embedded using a JB-4 embedding kit
(PolySciences, Warrington, Pa.) (Liu, et al., J. Bone Mineral Res.
5:973-82, 1990). When necessary, an alternative embedding method
(paraffin embedding) was used to speed up the embedding process.
Cross sections of calvariae cut at 5 .mu.m were obtained and
stained for tartrate-resistant acid phosphatase (TRAP) activity and
counterstained with methyl green and thionin for cell morphology
(Liu, et al., ibid.). Osteoclasts were identified by TRAP stain,
multinucleation, large cell size, and irregular cell shape. The
number of osteoclasts were counted from endocranial and ectocranial
bone surfaces and expressed as number/mm perimeter. The size of all
the osteoclasts counted was also measured using a Bone Morphometry
program (Liu, et al., ibid.; Bain, et al., J. Bone Miner. Res.
8:435-42, 1993). This histomorphometric method demonstrated
increases in the number and size of osteoclasts due to human
parathyroid hormone (PTH 1-34) treatment. This PTH-induced increase
was suppressed by treatment with human calcitonin.
Calcitonin mimetic compounds were evaluated in a similar fashion
for their ability to suppress PTH-induced increases in osteoclast
number and size (Table 2). Cell toxicity (or death) was also
evaluated by the appearance of pyknotic nuclei in a small number of
bone cells. With an increased level of toxicity, a further increase
in the number of these pyknotic nuclei, detachment of cells from
bone surfaces, and losses of cytoplasmic stain and cell boundaries
were observed. The osteocytic space also appeared empty.
TABLE 2: Effect of Calcitonin Mimetics on PTH-Induced Bone
Resorption in Mouse Calvariae Release of PTH Induced Ca++
Calcitonin Mimetic IC50 (.mu.g/ml) Histology 628-033 30 Decreases
in bone destruction by PTH, Oc size and # 628-035 23 Decreases in
bone destruction by PTH, Oc size and # 628-055 14 Decreases in bone
destruction by PTH, Oc size and #
For comparative purposes, the IC50 for human calcitonin is about
0.2 to 0.5 nM.
There was no apparent toxicity or tissue necrosis detected based on
histological observation of calvaria treated with up to 50 ug/ml of
compound.
Induction of Hypocalcemia in Rats
This assay is based on the in vivo acute effect of calcitonin on
osteoclasts, which causes rapid retraction of osteoclasts from bone
surface (typically within 30 minutes) and which results in
decreased bone resorption. See Mills, et al., in Endocrinology
1971--Proceedings of the Third International SymDosium, Taylor
(ed), Heinemann Medical, London, pp. 79-88 (1972) and Singer, et
al., Clin. Endocrinol. 5(Supp):333s-40s, 1976. The assay method was
modified from the method described by Sturtridge and Kumar, Lancet
545:725-6, 1968. For the assay of hypocalcemic activity, weanling
male Holtzman Sprague-Dawley rats (22 days old) are infused with
vehicle (PBS with 1 mM HCl and 0.1% BSA), calcitonin or calcitonin
mimetics through the tail vein. One hour later, blood samples are
collected by orbital sinus puncture to determine serum levels of
calcium. A decrease in serum calcium indicates a hypocalcemic
response. The hypocalcemic response is dose-dependent as determined
using salmon calcitonin (0.5, 2.5, 5, 50 and 100 ng/rat) in this
model. Inhibition of PTH-induced hypercalcemia in TPTX rats
Continuous PTH infusion is associated with extensive destruction
and severe hypercalcemia in thyroparathyroidectomized (TPTX) rats.
See Thompson, et al., Proc. Natl. Acad. Sci. USA 85:5673-7, 1988.
An animal model has been successfully established. See Liu, et al.,
J. Bone Mineral Res. 11 (Suppl. 1):S206, 1996. For in vivo assay,
male Sprague-Dawley rats (weighing about 150 g) are
thyroparathyroidectomized and the success of surgery is determined
by measuring the levels of serum calcium. Animals which are
successfully operated on (serum calcium levels less than 8 mg/dl)
are maintained on a low calcium diet (0.02% Ca and 0.6% P, ICN
special diet) and infused s.c. with vehicle (PBS with 1 mM HCl and
0.1% BSA), PTH (75 ug human PTH 1-34/kg body weight/day),
PTH+calcitonin (salmon calcitonin 50 U/kg body weight/day), or
PTH+calcitonin mimetic via Alzet osmotic minipumps (Model 1003D,
Alza Corp., Palo Alto, Calif.). Two days after infusion, animals
are sacrificed and blood samples are collected to determine if the
hypercalcemic response induced by PTH is inhibited by
co-administration of calcitonin or calcitonin mimetic.
Additionally, tibial and kidney samples are collected to determine
osteoclastic bone resorption and nephrocalcinosis, respectively,
and to confirm the findings in serum chemistry. Severe
hypercalcemia induced by PTH has been shown to be accompanied by
increases in the number and size of osteoclasts, extensive bone
destruction, and calcification in kidneys (nephrocalcinosis)
following only two days of treatment (see, Liu, et al., ibid.). The
serum, bone and kidney changes were attenuated by co-administration
of Conn.
Bone Loss Induced by Combined Ovariectomy and Immobilization in
Rats
Estrogen deficiency and immobilization both induce bone loss in
humans and in experimental animals. The combined effects cause
severe osteopenia. See, Strachan, et al., J. Bone Mineral Res.
11(Suppl. 1):S456, 1996. A few studies have also shown that
calcitonin is effective at reducing bone loss associated with
combined ovariectomy and immobilization. See, Hayashi, et al., Bone
10:25-8, 1989 and McSheehy, et al., Bone 16:435-44, 1995. Slightly
modified procedures were recently used to reproduce those results
and demonstrate that calcitonin is very effective at reducing bone
loss associated with the combined surgery, when evaluated by pQCT
or histomorphometry in rats (see, Strachan, et al., ibid.).
For induction of bone loss, 2-month old Sprague-Dawley rats
(weighing about 200 g) are ovariectomized and immobilized by
neurotomy of the sciatic nerve in the left hind limb. The
immobilized animals are treated with vehicle (PBS with 1 mM HCl and
0.1% BSA), calcitonin (15 U/kg body weight/day), or calcitonin
mimetics for 6 weeks. Calcein injections (15 mg/kg body weight/day)
are given i.p. at 9 and 2 days prior to sacrifice. Bone
histomorphometry is performed as previously described (see, Liu, et
al., J. Bone Mineral Res. 5:973-82, 1990) to determine the effects
of calcitonin and calcitonin mimetics. Calvarial Assay to Determine
Calcitonin Escape
Calvaria from 4-day old neonatal CD-1 mice (pregnant mice received
from Charles River Laboratories) are trimmed with fine-tipped
scissors to leave the parietal regions, including the sagittal
suture. These trimmed bones are placed singly per well into 6-well
culture cluster plates (Costar) with 1 ml/well of growth medium,
(Eagle's with Earle's salts (GIBCO BRL) containing 4.5 g/l glucose,
0.29 mg/ml L-glutamine, 1 mM sodium pyruvate, 15% heat-inactivated
horse serum, antibiotics (penicillin-G 50 .mu.g/ml, streptomycin 50
.mu.g/ml, and neomycin 100 .mu.g/ml) and 5 nM parathyroid hormone
(PTH) 1-34 (Sigma)), and rocked gently (RedRocker.TM.) at
37.degree. C. in a 5% CO.sub.2 humidified incubator for 17.5 hours
preincubation. The concentration of PTH is chosen to insure maximum
resorption.
Following preincubation, medium is removed and replaced with 1
ml/well of growth medium, as above, containing 20 or 30 .mu.g/ml of
the calcitonin mimetic in DMSO (final assay concentration of DMSO
less than or equal to 1%). Positive controls which can be used
include, growth medium containing 0.5, 1.0, 5.0 or 10 nM human
calcitonin (hCT) and/or 0.01, 0.02, 0.05 or 0.2 nM salmon
calcitonin (sCT). Control wells that do not receive PTH, human or
salmon calcitonin or the calcitonin mimetic. are included for
determination of calcium release from untreated bones. All control
wells contain a final assay concentration of DMSO equal to that
present in the calcitonin mimetic treated wells.
Five bones are included in each sample group. Bones are incubated
for 4, 8, 11, 24, 50.5, 72.5 and 98 hours. At each time point the
media is removed and fresh dilutions of compound in media are added
to the calvaria. After the media is removed, total calcium
measurements are made using a Nova 7/7+7 Electrolyte Analyzed (Nova
Biomedical) according to the manufacturer's specifications.
Induction of bone resorption by PTH is seen as an increase in the
concentration of calcium in the growth medium due to degradation of
the bone matrix.
Human and salmon calcitonin and biologically active calcitonin
mimetics inhibit the bone resorptive process as demonstrated by a
lowering of the calcium in growth medium as compared to bones
treated with PTH alone. The inhibitory effect of hCT and sCT is
lost after a period of time, generally 24 hours, and the rate of
resorption follows the same slope as that of PTH alone. Those
mimetic compounds which do not have this escape will be able to
continue inhibition of resorption for a longer period of time.
Calvaria can also be observed, as described above, at each time
point for signs of inhibition and signs of toxicity.
From the foregoing, it will be appreciated that, although specific
embodiments of the invention have been described herein for
purposes of illustration, various modifications may be made without
deviating from the spirit and scope of the invention. Accordingly,
the invention is not limited except as by the appended claims.
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